Automated fluorescence in situ hybridization detection of genetic abnormalities

ABSTRACT

Automated sample analysis is performed by a computer-implemented apparatus and method for distinguishing objects of interest in an optical field from other objects and background in the optical field, collectively called background. Once an object has been identified, the color comprised of a combination of the red, green and blue components of the pixels occupied by the image of the object of interest, or another parameter of interest relative to that object can be measured and stored. This computer-implemented analysis apparatus and method is performed on objects of interest in the sample which are tagged using fluorescent tags. The sample may be a cell sample containing a nucleic acid target and the tagging achieved by fluorescence in situ hybridization.

This application is a continuation of U.S. patent application Ser. No.08/689,562 filed Aug. 12, 1996, currently abandoned, which is acontinuation of U.S. patent application Ser. No. 08/316,778 filed Oct.3, 1994, currently abandoned.

FIELD OF THE INVENTION

The present invention relates to a computer-implemented method andapparatus for distinguishing objects of interest from other objects andbackground in an optical field. More particularly, the invention relatesto a computer-implemented method of identifying, characterizing andcounting objects in the optical field which are tagged usingfluorescence in situ hybridization to specifically visualize selectedobjects in the optical field. In particular, the invention relates tothe use of such a computer-implemented method and apparatus fordetermining genetic abnormalities.

BACKGROUND OF THE INVENTION

The normal human complement of chromosomes consists of the sexchromosomes (designated X and Y) and 22 autosomes (numbered 1-22). Ithas been estimated that a minimum of 1 in 10 human conceptions has achromosome abnormality. As a general rule, an abnormal number of sexchromosomes is not lethal, although infertility can result. In contrast,an abnormal number of autosomes typically results in early death. Of thethree autosomal trisomies found in live-born babies (trisomy 21, 18 and13), only individuals with trisomy 21 (more commonly known as Downsyndrome), survive past infancy.

Although Down syndrome is easily diagnosed after birth, prenataldiagnosis is problematic. To date, karyotyping of fetal cells remainsthe established method for the diagnosis of Down syndrome and othergenetic abnormalities associated with an aberration in chromosomalnumber and/or arrangement. Such genetic abnormalities include, forexample, chromosomal additions, deletions, amplifications,translocations and rearrangements. The assessment of such abnormalitiesis made with respect to the chromosomes of a healthy individual, i.e.,an individual having the above-described normal complement of humanchromosomes.

Genetic abnormalities include the above-noted trisomies, such as Downsyndrome, as well as monosomies and disomies. Genetic abnormalities alsoinclude additions and/or deletions of whole chromosomes and/orchromosome segments. Alterations such as these have been reported to bepresent in many malignant tumors. Thus, aberrations in chromosome numberand/or distribution (e.g., rearrangements, translocations) represent amajor cause of mental retardation and malformation syndromes (du Manoiret al., et al., Human Genetics 90(6): 590-610 (1993)) and possibly,oncogenesis. See also,. e.g., (Harrison's Principles of InternalMedicine, 12th edition, ed. Wilson et al., McGraw Hill, N.Y., N.Y., pp.24-46 (1991 )), for a partial list of human genetic diseases that havebeen mapped to specific chromosomes, and in particular, for a list of Xchromosome linked disorders. In view of the growing number of geneticdisorders associated with chromosomal aberrations, various attempts havebeen reported in connection with developing simple, accurate, automatedassays for genetic abnormality assessment.

In general, karyotyping is used to diagnose genetic abnormalities thatare based upon additions, deletions, amplifications, translocations andrearrangements of an individual's nucleic acid. The "karyotype" refersto the number and structure of the chromosomes of an individual.Typically, the individual's karyotype is obtained by, for example,culturing the individual's peripheral blood lymphocytes until activecell proliferation occurs, preparing single, proliferating (e.g.metaphase, and possibly, interphase) cells for chromosome visualization,fixing the cells to a solid support and subjecting the fixed cells to insitu hybridization to specifically visualize discrete portions of theindividual's chromosomes.

The rapid development of non-isotopic in situ hybridization techniquesand the general availability of an ever-expanding repertoire ofchromosome-specific DNA probes have extended the number of geneticdisorders for which karyotyping is feasible. See, e.g., Lichter et al.,"Analysis of Genes and Chromosomes by Non-isotopic in situHybridization", GATA 8(1): 24-35 (1991). Such methods include the use ofprobe sets directed to chromosome painting for visualizing one or morepreselected chromosomal subregions in a targeted fashion. Methods suchas these require at least a modicum of knowledge regarding the types ofaberration(s) expected in order to select useful DNA probescomplementary to target nucleic acids present in a clinical or tumorcell sample.

Nucleic acid hybridization techniques are based upon the ability of asingle stranded oligonucleotide probe to base-pair, i.e., hybridize,with a complementary nucleic acid strand. Exemplary in situhybridization procedures are disclosed in U.S. Pat. No. 5,225,326 andcopending U.S. patent application Ser. No. 07/668,751, the entirecontents of which are incorporated herein by reference. Fluorescence insitu hybridization ("FISH") techniques, in which the nucleic acid probesare labeled with a fluorophor (i.e., a fluorescent tag or label thatfluoresces when excited with light of a particular wavelength),represents a powerful tool for the analysis of numerical, as well asstructural aberrations chromosomal aberrations. See, e.g., PCTApplication WO 94/02646, inventors M. Asgari et al., published Feb. 3,1994, (hereinafter, "Asgari") co-pending U.S. patent application Ser.No. 07/915,965; P. Lichter, et al., Genet. Anal. Tech. Appl. 8: 24-35(1991); and S. Du Manoir, et al., Human Genetics 90(6): 590-610 (1993),the entire contents of which publications are incorporated herein byreference.

Asgari reports in situ hybridization assays for determining the sex of afetus, genetic characteristics or abnormalities, infectious agents andthe like, by nucleic acid hybridization of fetal cells such as thosecirculating in material blood. The fetal cells are distinguished frommaternal cells present in the fixed sample by staining with an antibodywhich specifically recognizes the maternal or fetal cell or by in situhybridization to detect one or more fetal mRNAs. The method reportedlyis useful for detecting chromosomal abnormalities in fetal cells.However, the fetal cells must be enriched prior to analysis.

PCT Application WO 94/02830, inventors M. Greaves, et al., publishedFeb. 3, 1994, (hereinafter, "Greaves") report a method for phenotypingand genotyping a cell sample. The method involves contacting a fixedcell with an antibody labeled with a first fluorophor for phenotypingthe cell via histochemical staining, followed by contacting the fixedcell with a DNA probe labeled with a second fluorophor for genotypingthe cell. The first and second fluorophors fluoresce at differentwavelengths from one another, thereby allowing the phenotypic andgenetic analysis on the identical fixed sample.

Despite the above-described advances in the development of fluorescentin situ hybridization methods for the diagnosis of geneticabnormalities, the analysis of the fluorophor-labeled sample remainslabor-intensive and involves a significant level of subjectivity. Thisis particularly true in connection with the prenatal diagnosis ofgenetic abnormalities in which fetal cells must either be isolated frommaternal cells or visually distinguished therefrom prior to assessmentfor genetic abnormalities. Thus, for example, a laboratory technicianmust manually prepare and sequentially stain the sample (first, with ahistochemical stain to phenotype the cells, second, with a hybridizationprobe to genotype the cell); visually select fetal cells from othercells in the optical field (using, for example, the above-mentionedhistochemical staining procedure); assess the relative distribution offluorescent color that is attributable to hybridization of thefluorophor-tagged probe; and compare the visually-perceived distributionto that observed in control samples containing a normal human chromosomecomplement. As will be readily apparent, the above-described procedureis quite time-consuming. Moreover, because the results arevisually-perceived, the frequency of erroneous results can vary from oneexperiment to the next, as well as from one observer to the next.

SUMMARY OF THE INVENTION

The instant invention overcomes these and other problems by providingcomputer-implemented methods for determining genetic abnormality whichthereby eliminate subjective analysis of selectively stainedchromosomes. More specifically, a method for detecting whether a geneticabnormality is present in a fixed sample containing at least one targetnucleic acid is provided. The method involves (i) receiving a digitizedcolor image of the fixed sample, which has been subjected tofluorescence in situ hybridization under conditions to specificallyhybridize a fluorophor-labeled probe to the target nucleic acid; (ii)processing the color image in a computer to separate objects of interestfrom background in the color image; (iii) measuring parameters of theobjects of interest so as to enumerate objects having specificcharacteristics; and (iv) analyzing the enumeration of objects withrespect to a statistically expected enumeration to determine the geneticabnormality. The numerical distribution of target nucleic acid isindicative of the genetic abnormality. The method is useful fordiagnosing genetic abnormalities associated with an aberration inchromosomal number and/or arrangement. Such "genetic abnormalities"include, for example, chromosomal additions, deletions, amplifications,translocations and rearrangements with respect to the chromosomes of ahealthy individual, i.e., an individual having the above-describednormal complement of human chromosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be discussed in connectionwith the figures. Like reference numerals indicate like elements in thefigures, in which:

FIG. 1 is a block diagram of one embodiment of the apparatus of theinvention;

FIG. 2 is a flowchart of the calibration steps of one embodiment of theinvention;

FIG. 3 is a flowchart of the preprocessing steps of one embodiment ofthe invention;

FIG. 4 is an illustration of a microscope slide showing an area ofobservation; and

FIGS. 5A and 5B are a flowchart of the main processing steps of oneembodiment of the invention.

DETAILED DESCRIPTION

A method for detecting whether a genetic abnormality is present in afixed sample is disclosed herein. The method involves (i) receiving adigitized color image of the fixed sample, which has been subjected tofluorescence in situ hybridization under conditions to specificallyhybridize a fluorophor-labeled probe to the target nucleic acid; (ii)processing the color image in a computer to separate objects of interestfrom background in the color image; (iii) measuring parameters of theobjects of interest so as to enumerate objects having specificcharacteristics; and (iv) analyzing the enumeration of objects withrespect to a statistically expected enumeration to determine the geneticabnormality. The method is useful for diagnosing genetic abnormalitiesassociated with an aberration in chromosomal number and/or arrangement.Thus, for example, the invention can be used to detect chromosomalrearrangements by using a combination of labeled probes which detect therearranged chromosome segment and the chromosome into which the segmentis translocated.

As used herein, "genetic abnormalities" refers to an aberration in thenumber and/or arrangement of one or more chromosomes with respect to thecorresponding number and/or arrangement of chromosomes obtained from ahealthy subject, i.e., an individual having a normal chromosomecomplement. Genetic abnormalities include, for example, chromosomaladditions, deletions, amplifications, translocations and rearrangementsthat are characterized by nucleotide sequences of as few as about 15base pairs and as large as an entire chromosome.

The method is useful for determining one or more genetic abnormalitiesin a fixed sample, i.e., a sample attached to a solid support which hasbeen treated in a manner to preserve the structural integrity of thecellular and subcellular components contained therein. Methods forfixing a cell containing sample to a solid support, e.g., a glass slide,are well known to those of ordinary skill in the art.

The sample contains at least one target nucleic acid, the distributionof which is indicative of the genetic abnormality. By "distribution", itis meant the presence, absence, relative amount and/or relative locationin one or more nucleic acids (e.g., chromosomes) known to include thetarget nucleic acid. In a particularly preferred embodiment, the targetnucleic acid is indicative of a trisomy 21 and thus, the method isuseful for diagnosing Down syndrome. In a particularly preferredembodiment, the sample intended for Down syndrome analysis is derivedfrom maternal peripheral blood. More particularly, lymphocytes areisolated from peripheral blood according to standard procedures, thecells are attached to a solid support (e.g., by centrifuging onto glassslides), and fixed thereto according to standard procedures (see, e.g.,the Examples) to permit detection of the target nucleic acid.

Fluorescence in situ hybridization refers to a nucleic acidhybridization technique which employs a fluorophor-labeled probe tospecifically hybridize to and thereby, facilitate visualization of, atarget nucleic acid. Such methods are well known to those of ordinaryskill in the art and are disclosed, for example, in U.S. Pat. No.5,225,326; U.S. patent application Ser. No. 07/668,751; PCT WO 94/02646,the entire contents of which are incorporated herein by reference. Ingeneral, in situ hybridization is useful for determining thedistribution of a nucleic acid in a nucleic acid-containing sample suchas is contained in, for example, tissues at the single cell level. Suchtechniques have been used for karyotyping applications, as well as fordetecting the presence, absence and/or arrangement of specific genescontained in a cell. However, for karyotyping, the cells in the sampletypically are allowed to proliferate until metaphase (or interphase) toobtain a "metaphase-spread" prior to attaching the cells to a solidsupport for performance of the in situ hybridization reaction.

Briefly, fluorescence in situ hybridization involves fixing the sampleto a solid support and preserving the structural integrity of thecomponents contained therein by contacting the sample with a mediumcontaining at least a precipitating agent and/or a cross-linking agent.Exemplary agents useful for "fixing" the sample are described in theExamples. Alternative fixatives are well known to those of ordinaryskill in the art and are described, for example, in the above-notedpatents and/or patent publications.

In situ hybridization is performed by denaturing the target nucleic acidso that it is capable of hybridizing to a complementary probe containedin a hybridization solution. The fixed sample may be concurrently orsequentially contacted with the denaturant and the hybridizationsolution. Thus, in a particularly preferred embodiment, the fixed sampleis contacted with a hybridization solution which contains the denaturantand at least one oligonucleotide probe. The probe has a nucleotidesequence at least substantially complementary to the nucleotide sequenceof the target nucleic acid. According to standard practice forperforming fluorescence in situ hybridization, the hybridizationsolution optionally contains one or more of a hybrid stabilizing agent,a buffering agent and a selective membrane pore-forming agent.Optimization of the hybridization conditions for achieving hybridizationof a particular probe to a particular target nucleic acid is well withinthe level of the person of ordinary skill in the art.

In reference to a probe, the phrase "substantially complementary" refersto an amount of complementarity that is sufficient to achieve thepurposes of the invention, i.e., that is sufficient to permit specifichybridization of the probe to the nucleic acid target while not allowingassociation of the probe to non-target nucleic acid sequences under thehybridization conditions employed for practicing the invention. Suchconditions are known to those of ordinary skill in the art of in situhybridization.

The genetic abnormalities for which the invention is useful are thosefor which there is an aberration in the number and/or arrangement of oneor more chromosomes with respect chromosomes obtained from an individualhaving a normal chromosome complement. Exemplary chromosomes that can bedetected by the present invention include the human X chromosome, the Ychromosome and chromosomes 13, 18 and 21. For example, the targetnucleic acid can be an entire chromosome, e.g., chromosome 21, whereinthe presence of three copies of the chromosome ("the distribution" ofthe target nucleic acid) is indicative of the genetic abnormality, Downsyndrome). Exemplary probes that are useful for specifically hybridizingto the target nucleic acid (e.g. chromosome) are probes which can belocated to a chromosome(s) that is diagnostic of a genetic abnormality.See e.g., Harrison's Principles of Internal Medicine, 12th edition, ed.Wilson et al., McGraw Hill, N.Y., N.Y. (1991).

The preferred embodiment of the invention is directed to the prenataldiagnosis of Down syndrome by detecting trisomy 21 (discussed below) infetal cells present in, for example, maternal peripheral blood,placental tissue, chorionic villi, amniotic fluid and embryonic tissue.However, the method of the invention is not limited to analysis of fetalcells. Thus, for example, cells containing the target nucleic acid maybe eukaryotic cells (e.g., human cells, including cells derived fromblood, skin, lung, and including normal as well as tumor sources);prokaryotic cells (e.g., bacteria) and plant cells. According to oneembodiment, the invention is used to distinguish various strains ofviruses. According to this embodiment, the target nucleic acid may be ina non-enveloped virus or an enveloped virus (having a non-envelopedmembrane such as a lipid protein membrane). See, e.g., Asgari supra.Exemplary viruses that can be detected by the present invention includea human immunodeficiency virus, hepatitis virus and herpes virus.

The oligonucleotide probe is labeled with a fluorophor (fluorescent"tag" or "label") according to standard practice. The fluorophor can bedirectly attached to the probe (i.e., a covalent bond) or indirectlyattached thereto (e.g., biotin can be attached to the probe and thefluorophor can be covalently attached to avidin; the biotin-labeledprobe and the fluorophor-labeled avidin can form a complex which canfunction as the fluorophor-labeled probe in the method of theinvention).

Fluorophors that can be used in accordance with the method and apparatusof the invention are well known to those of ordinary skill in the art.These include 4,6-diamidino-2-phenylindole (DIPA), fluoresceinisothiocyanate (FITC) and rhodamine. See, e.g., the Example. See alsoU.S. Pat. No. 4,373,932, issued Feb. 15, 1983 to Gribnau et al., thecontents of which are incorporated herein by reference, for a list ofexemplary fluorophors that can be used in accordance with the methods ofthe invention. The existence of fluorophors having different excitationand emission spectrums from one another permits the simultaneousvisualization of more than one target nucleic acid in a single fixedsample. As discussed below, exemplary pairs of fluorophors can be usedto simultaneously visualize two different nucleic acid targets in thesame fixed sample.

The distribution of the target nucleic acid is indicative of the geneticabnormality. See e.g., Asgari supra. The genetic abnormalities of theinvention include deletions, additions, amplifications, translocationsand rearrangements. For example, a deletion is identified by detectingthe absence of the fluorescent signal in the optical field. To detect adeletion of a genetic sequence, a population of probes are prepared thatare complementary to a target nucleic acid which is present in a normalcell but absent in an abnormal one. If the probe(s) hybridize to thenucleic acid in the fixed sample, the sequence will be detected and thecell will be designated normal with respect to that sequence. However,if the probes fail to hybridize to the fixed sample, the signal will notbe detected and the cell will be designated as abnormal with respect tothat sequence. Appropriate controls are included in the in situhybridization reaction in accordance with standard practice known tothose of ordinary skill in the art.

A genetic abnormality associated with an addition of a target nucleicacid can be identified, for example, by detecting binding of afluorophor-labeled probe to a polynucleotide repeat segment of achromosome (the target nucleic acid). To detect an addition of a geneticsequence (e.g., trisomy 21), a population of probes are prepared thatare complementary to the target nucleic acid. Hybridization of thelabeled probe to a fixed cell containing three copies of chromosome 21will be indicated as discussed in the Examples.

Amplifications, translocations and rearrangements are identified byselecting a probe which can specifically bind to the nucleic acid targetfor which amplification, translocation or rearrangement is suspected andperforming the above-described procedures. In this manner, a fluorescentsignal can be attributed to the target nucleic acid which, in turn, canbe used to indicate the presence or absence of the genetic abnormalityin the sample being tested.

Each of the above-identified patents, patent publications and referencesis incorporated in its entirety herein by reference.

EXAMPLES

Sample Preparation

(1) Slide Preparation

Fresh lymphoblasts were obtained from patients according to standardprocedures known to those of ordinary skill in the art. In general,between about 10 and about 20 ml of peripheral maternal blood is usedfor analysis. Twenty mls of maternal blood (containing about 100 fetalcells) was centrifuged onto glass slides, for 5 minutes at 5000 rpm. Theslides were dipped in chilled 80% ethanol/water (v/v) for 5 minutes, airdried and stored. Alternatively, other useful fixatives can be used,including, e.g., acetic acid, methanol, acetone, and combinationsthereof, for example ethanol/methanol mixture 3:1 (v/v).

(2) Fluorescence in Situ Hybridization

Slides were stored for up to two weeks at room temperature beforehybridization. Cell preparations were heat denatured in 2× SSC (0.3 MNaCl, 30 mM Na citrate)-70% formamide at 70° C. for two minutes,dehydrated through 70%, 90% and 100% alcohol and air dried at roomtemperature. Hybridization buffer (50% formamide/10% dextran sulphate in2× SSC pH 7.0) containing the probe was denatured at 70° C. for sevenminutes and cooled on ice at 4° C. according to standard hybridizationprocedures. An aliquot of the hybridization solution was added to eachslide under a sealed coverslip and incubated overnight in a moistchamber at 37° C. The coverslips were removed in 2× SSC and the slideswashed in 50% formamide/2× SSC (pH 7.0) at 45° C. for forty minutes,twice in 2× SSC (pH 7.0) for five minutes at room temperature and oncein 1× SSC (pH 7.0) for five minutes at room temperature. Slides werestored at room temperature in phosphate buffer (0.1 M NaH₂ PO₄, 0.1 MNa₂ HPO₄ pH 8.0).

Detection: When fluoroscein is the probe dye, the dye is first excitedwith light having a wavelength of 488 nm and then the emitted light ismeasured. For the emitted light, a 540 bp filter is used, i.e., onlylight with a wavelength between 520 and 560 nm is allowed to pass. Themethod of detection and the apparatus used therefor are disclosed here.At this point, it is noted that the emitted light is filtered beforearriving at a detection apparatus or a human observer. Such filtering isa standard procedure of florescence microscopy. This pre-detectionfiltering is not specifically part of the image processing methods ofthe present invention, but is rather a conventional preprocessing step.

PREFERRED EMBODIMENT

Apparatus

An apparatus according to one embodiment of the present invention is nowdescribed in connection with FIG. 1. FIG. 1 shows the basic elements ofa system according to this embodiment. The basic elements of the systeminclude an X-Y stage 101, a mercury light source 103, a fluorescencemicroscope 105, a color, chilled-CCD camera 107, a personal computer(PC) 109, and one or more monitors, e.g. 111 and 113. The individualelements of the system may be custom-built or purchased off-the-shelf asstandard components. These elements will now be described in somewhatgreater detail.

The X-Y stage 101, is a positionable stage suitable for use withmicroscope 105. For example, X-Y stage 101 may be a manual stage havingconventional micrometer adjustments for positioning a microscope slide,by a human operator. Alternatively, X-Y stage 101 may be controlled bythe PC 109, using a software program executing in the PC. Such aPC-controlled X-Y stage 101 may therefore include a stage controllercircuit card 101, plugged into an expansion bus of the personal computer109. Electronically controlled stages such as described here areproduced by microscope manufacturers, including Olympus (Tokyo, Japan)as well as other manufacturing concerns.

The microscope 105 may be any fluorescence microscope equipped with areflected light fluorescence illuminator and suitable lenses. Forexample, the microscope is preferably equipped with an oil immersion100× objective lens, providing a total magnification between about 600×and 1000×. It is known that fluorescence images obtained by such amicroscope have a decreasing light intensity from the center to theboundary of the optical field. The mercury light source 103 is thereforecapable of providing consistent and substantially uniform illuminationof a sample which produces a fluorescence image viewed through themicroscope 105.

The image is viewed by camera 107. The camera 107 is a color, chilled,3-chip CCD camera providing high sensitivity and resolution. Chilling ofthe CCD, for example, thermo-electric chilling, minimizes the darkcurrent generated during long time exposure, i.e., it increased thesignal-to-noise ratio of the CCD. This enables extremely high qualityimages to be captured under low illumination and exposure times as longas 5 seconds. A suitable camera is a HAMAMATSU (HAMAMATSU PHOTONICSK.K., HAMAMATSU CITY, JAPAN, model C5820-10). The Hamamatsu camera is aPAL, color, chilled-CCD camera with a resolution of 739×512 pixelscapable of exposure times up to 10 seconds. The output of camera 107 isfed to a frame grabber and image processor circuit 117 installed in thePC 109. Of course, other cameras and camera technologies may be suitableif sufficient sensitivity and resolution are available to obtain imageswhich may be processed as discussed below.

The frame grabber and image processor circuit 117 may be, for example, acombination of a MATROX IM-CLD color image capture module and a MATROXIM-640 image processing module, featuring hardware-supported imageprocessing capabilities. Thus, it executes SIMPLE-based softwareinstructions high speeds, because the SIMPLE software program, availablefrom COMPIX, INC. IMAGING SYSTEMS (Mars, Pa.), has been designed to takeadvantage of the MATROX hardware capabilities. The MATROX boards supporta dedicated SVGA monitor 113. Any SVGA monitor suitable for use with theimage processing boards can be used. In this embodiment, the dedicatedmonitor 113 is a ViewSonic 4E SVGA monitor, from ViewSonic (Walnut,Calif.).

In order to have sufficient processing and storage capabilitiesavailable, it is preferred that the PC 109 be an INTEL 486-based PChaving at least 8 MB RAM and at least 200 MB of hard disk drive storagespace (INTEL is a registered trademark of the Intel Corporation). The PC109 of the present embodiment further includes a monitor 111. Other thanthe specific features described herein the PC 109 is conventional, andcan include keyboard, printer or other desired peripheral devices notshown. Also, PCs of greater or lesser specifications may be used, with acommensurate increase or decrease in capability to be expected.

Method

The PC 109 executes a software program called SIMPLE which controlsoperation of the frame grabber and image processor circuit 117. SIMPLEalso processes images captured by frame grabber and image processorcircuit 117 and subsequently stores images and processed data in PC 109as disk files. SIMPLE provides an icon-based environment withspecialized routines particularly suitable for performing such imageprocessing tasks as filtering, object selection and measurement. Most ofthe SIMPLE tasks are directed by a human operator using a pointingdevice connected to PC 109, such as a mouse or trackball (not shown).

In order to process images using SIMPLE, a number of image calibrationsteps must first be taken.

The flowchart of FIG. 2 shows the calibration steps of this embodimentof the present invention. Calibration modifies parameters of thesoftware program to compensate for day to day variation in systemperformance, as well as variations from one microscope 105, camera 107,and other system components to another.

In particular, calibration is directed to determining imagemagnification so that accurate size measurements may be made. Objectsize measurements in SIMPLE are initially made in pixels. The operatorcan calibrate the image using a distance calibration standard such as amicroscope graticule or other solid support, e.g., a culture plate or awell having known, fixed distances marked thereon, as follows. In step201, the SIMPLE Image Capture utility is loaded into the PC 109 forexecution. An image of a microscope graticule slide (or other distancecalibration standard) is then grabbed, step 203, using the microscope105 and the camera 107 using a specific, nominal total magnification,e.g., a 10× ocular and a 40× objective lens. This image is processed inthe processing board. Selecting the SIMPLE CALIBRATE function, step 205,causes a cursor to appear on the image monitor. The cursor is moved inresponse to operator input made using the pointing device to the startof the known distance and that point designated by the operator. Thecursor is then moved, causing the SIMPLE software program to draw arubber-band like line which the operator then joins to the other end ofthe known distance, whereupon that point is also designated. Then theoperator answers the question "How long is the line" by entering thenumber of calibration units indicated by the line. A calibration factoris thus updated, step 207. The MEASURE function may now be selected tocheck the calibration, step 209. Selecting the function will cause thecursor to again appear on the monitor. The cursor is moved to the firstpoint of a distance to be measured, which is designated, then therubber-band line is dragged to the other end of the distance to bemeasured, which is designated. The measured distance is verified, step211. If the measured distance does not match the actual distance, thencontrol may be returned to step 205 to re-calibrate. Otherwise, thecurrent calibration is saved by selecting a DISK SAVE function, step213. Multiple calibration files can be saved to be used in futureapplications, for example employing different lens combinations.

The automated analysis employs two principal procedures: a preprocessingprocedure and a main procedure. Except where noted, the steps of theprocedures are performed by the computer executing SIMPLE softwareinstructions to carry out the functions named. The functions named areavailable directly in simple, as named commands.

A new slide properly stained using the fluorescence in situhybridization (FISH) technique is placed under the fluorescencemicroscope. The objects of interest which are to be recognized, i.e.,the nuclear or chromosomal areas, have specific chromatic features.Multiple targets can be delineated simultaneously in a particularspecimen by combining fluorescence detection procedures. That is, ifdifferent targets are labeled with different fluorophors that fluoresceat different wavelengths, then the software program can be made toseparately identify objects emitting the different fluorophors, providedfull color information is available in the image. Targets with differingaffinities for different fluorophors may be differentiated by the colorcombinations emitted. Each target may emit at wavelengths correspondingto two or more fluorophors, but the intensity of each may differ, forexample. Thus, all three color components of the microscopic images areused during processing.

For each new specimen inserted under the microscope, a preprocessingprocedure is first executed. The flowchart of FIG. 3 shows thepreprocessing steps of this embodiment of the present invention.Preprocessing permits the software to compensate forspecimen-to-specimen variations.

Preprocessing

Preprocessing produces a number of results which are required bysubsequent steps performed as part of the main processing steps. Theseresults are passed from the preprocessing steps to the main processingsteps by any conventional means, such as storing them in RAM or on disk.

A microscope image is first grabbed by the frame grabber and imageprocessing circuits (FIG. 1, 117), step 301. The specimen has beenprepared and placed in the optical path of the microscope in such a waythat the image grabbed includes one or more interphase nuclei or amixture of interphase nuclei and metaphase chromosomes. Next, a regionof interest (ROI) is manually selected using the pointing device, step303, to include intracellular, cytoplasmic and nuclear image portions,but to exclude fluorescent areas of the nucleus, i.e. interestingobjects. For the regions not of interest which have been selected,maximum and minimum values of the red, green and blue components of thepixels of those regions are determined. The maximum and minimum red,green and blue values 305 are then passed to the main processing steps.In step 307, high and low area limits are set which define the largestand smallest chromosomal areas to be recognized. The high and low arealimits are placed on a qualifier list 309, which is also passed to themain processing steps. Also placed on the qualifier list 309 is anindication or flag that the remove edge objects function has been set,step 311. In subsequent processing, the remove edge objects functionwill cause objects whose boundaries intersect the edge of the image tobe removed from the regions to be considered. The parameters of selectedobjects which are to be measured are then selected, step 313, and placedon parameter list 315. Parameter list 315 is also passed to the mainprocessing steps. Finally, the operator selects a name for an ASCIIresult file in which parameter measurements are saved, step 317. Thefile name, 319, is passed to the main processing steps.

Parameters which may be selected to be measured and placed on parameterlist 315 may include such values of interest as the area of the objectand the mean red, green and blue intensity values within the object. Bychoice of parameters to be placed on parameter list 315 and the valuesof qualifiers placed on qualifier list 309. The present invention may beapplied to the taking of other measurements or the making of otherdeterminations than those to which the specific embodiment described hasbeen applied. For example, by changing the high and low area limitsplaced on qualifier list 309, objects of different sizes than the sizeof a chromosome may be detected. Likewise, the parameters noted inconnection with the present embodiment are suitable for distinguishingbetween particular fluorescent labels applied to particular chromosomesand which fluoresce at different wavelengths. However, other parameterscould be measured which are suitable for making other determinationsconcerning the detected image.

After preprocessing, the system performs the automated analysis by therepeated execution of the main procedure.

The slide containing the FISH-treated cells is positioned into the X-Ystage 101. The X-Y stage 101 is moved to an initial observation position(FIG. 4, 401). A processing loop is executed repeatedly until either apredetermined number of cells of a particular type have been identifiedand measured or an entire area of best observation (FIG. 4, A) has beenanalyzed. In the application for which the present embodiment isintended, identifying multiple targets of chromosomal DNA, the loopwould preferably be executed until 100 nuclei have been processed. Eachnucleus is manually selected in the successive optical fields. Datarepresenting the measurement of the chromosomal areas within thosenuclei are collected in an ASCII file.

Execution of the processing loop is controlled by the operator whoselects the nuclear areas to be processed through manual delineation ofa wider area containing the nucleus, i.e., a wide Region Of Interest(ROI) containing only the nuclear area in which the chromosomal areasare to be counted.

The computer instructions defining the main processing procedure arecontained in a "work" file which is executed automatically. Theinstructions include instructions to suspend execution for the operatorto select the Region of Interest containing the nucleus. The mainprocessing procedure is now described in detail.

Main Procedure

Using the apparatus described above in connection with the SIMPLEsoftware system, the main processing steps first grab an image of theprepared slide, step 501, which includes at least one nucleus containingfluorescing regions. An ROI containing a single nucleus havingfluorescence regions is then selected, step 503. The selection isconventionally performed using the pointing device. In step 505, theimage is automatically processed to select the background, ornon-interesting portions of the ROI, by using the maximum and minimumred, green and blue values 305 passed from the preprocessing procedure,step 505. That is, those pixels whose red, green and blue intensityvalues fall between the maximum and minimum values obtained inpreprocessing are selected as part of the background. At this point, thebackground is selected and the objects of interest are not selected.Therefore, a logical NOT operator is applied to the selection, causingthe objects to be selected rather than the background, step 507. A setof complex filtering steps, steps 5000, described below, are applied togenerate a final selection of those areas considered interesting, inthat they contain the fluorescent objects desired to be measured. Thequalifier list 309 is then applied against the characteristics of theobjects remaining, to eliminate objects outside the high and low arealimits and also to eliminate those objects on the edges of the region ofinterest, step 509. The parameters contained on parameter list 315 arethen measured at step 511, and the results stored, step 513, in a resultfile 515 having the file name 319 determined by the preprocessing steps.At this point, either the operator or a counter in the software programdetermines whether the main processing steps have been performed asufficient number of times, and main processing is done, step 517. If itis determined that additional passes through the main processing stepsare required, then control passes to step 501, wherein a new image isgrabbed.

The above-described main processing steps are repeated until astatistically significant number of samples have been measured. Forexample, in order to detect the genetic abnormality of a trisome ofchromosome 21, 100 cell nuclei should be measured, requiring 100 passesthrough the main processing steps.

The filtering steps 5000 operate on a pixel-by-pixel basis, as follows.In step 5001, a hole filling filter is applied to the image. Thisfilter, available through the SIMPLE language, determines when darkholes have appeared within the lighter fluorescent chromosomes bysearching for dark areas within light objects. Those areas are lightenedup. The output of the hole filling filter is held in a temporary imagefile 5101, as well as being used as the input to the erosion filter,step 5003. Erosion filtering, also available through the SIMPLElanguage, replaces the center pixel of a small kernel with the darkestpixel in the kernel. In the preferred embodiment, the kernel used is3×3. A separate operation, step 5005 is next performed, to grow theobjects until they meet, but do not merge. This step also createsoutlines, defining the edges of all the objects. A logical NOToperation, step 5007, causes the pixels within the outlines to becomeselected rather than the outlines. Finally, in step 5009, the result ofstep 5007 is logically ANDed with the stored temporary image file 5101.This causes only those pixels which are defined in both the temporaryimage file 5101 and the output of step 5007 to be retained.

If a combination of fluorescence detection procedures is used, more thantwo chromosomal areas may be detected per nucleus. Therefore, it ispossible to recognize two chromosomal areas relative to chromosomes 21,another two relative to chromosome 18, one relative to chromosome X andone relative to chromosome Y, enabling the discovery of possiblenumerical aberrations detected by the enumeration of hybridizationsignals. The enumeration of the hybridization signals is executed aftercompleting the measurement of 100 nuclei through an application programexternal to SIMPLE, compiled using CLIPPER (COMPUTER ASSOCIATES, CA).This program reads the measurement results ASCII file and classifies thechromosomal areas detected according to their RGB color combination.When two or more different fluorophors are used in combination,different combinations of RGB color values may be used to distinguishdifferent targets, some targets of which may be labeled by more than onefluorophor. For example, targets may be stained with red and greenfluorophors, but one target may receive fluorophors to emit 30% red and70% green, another target may receive fluorophors to emit 70% red and30% green, while a third target may receive fluorophors to emit onlyred. The three targets may be distinguished on the basis of theirrelative emissions. If the number of signals indicative of a chromosomalarea corresponding to a specific chromosome, e.g., chromosome 21, isgreater than two to an operator-selected statistically significantlevel, then a report is issued identifying an increased likelihood fortrisomy 21 in the specific sample.

Although the present invention has been described in connection with theclinical detection of chromosomal abnormalities in a cell-containingsample, the image processing methods disclosed herein has other clinicalapplications. For example, the image processing steps described can beused to automate a urinalysis process. When the techniques of thepresent application are combined with those of application Ser. No.08/132,804, filed Oct. 7, 1993, a wide variety of cell types can bevisualized and analyzed, based on their morphology. Cell morphology canbe observed for the purpose of diagnosing conditions for which cellmorphology has been correlated to a physiological condition. Suchconditions are known to those of skill in the art. See, e.g., Harrison,supra. Various cell characteristics and abnormalities may be detectedbased on these techniques. Finally, it should be noted that theparticular source of the sample is not a limitation of the presentinvention, as the sample may be derived from a blood sample, a serumsample, a urine sample or a cell sample from the uterine cervix. Thecell visualization and image analysis techniques described herein may beused for any condition detectable by analysis of individual cells,either by morphology or other characteristics of the isolated cells.

The present invention has now been described in connection with a numberof specific embodiments thereof. However, numerous modifications whichare contemplated as falling within the scope of the present inventionshould now be apparent to those skilled in the art. Therefore, it isintended that the scope of the present invention be limited only by thescope of the claims appended hereto.

What is claimed is:
 1. A computer software product comprising a computerreadable storage medium having fixed therein a sequence of computerinstructions directing a computer system to detect whether a geneticabnormality is present in a fixed sample containing at least one targetnucleic acid, the instructions directing steps of:receiving a digitizedcolor image of the fixed sample, which has been subjected tofluorescence in situ hybridization under conditions to specificallyhybridize a fluorophor-labeled probe to the target nucleic acid;processing the color image in a computer to separate objects of interestfrom background; measuring size and color parameters of the revisedobjects of interest so as to identify and enumerate objects havingspecific predetermined characteristics associated with the targetnucleic acid; and analyzing the enumeration of objects with respect to astatistically expected enumeration to detect whether the geneticabnormality is present wherein the step of processing comprises thesteps of:passing the color image through a hole filling filter; passingthe filled color image through an erosion filter; operating on theeroded filled color image to define outlines around areas; selectingpixels within the outlines by performing a logical NOT operation; andperforming a logical AND operation between the selected pixels and thefilled color image.
 2. A method of operating a computer system to detectwhether a genetic abnormality is present in a fixed sample containing atleast one target nucleic acid, the method comprising:receiving adigitized color image of the fixed sample, which has been subjected tofluorescence in situ hybridization under conditions to specificallyhybridize a fluorophor-labeled probe to the target nucleic acid;processing the color image in a computer to separate objects of interestfrom background; measuring size and color parameters of the objects ofinterest so as to identity and enumerate objects having specificpredetermined characteristics associated with the target nucleic acid;and analyzing the enumeration of objects with respect to a statisticallyexpected enumeration to detect whether the genetic abnormality ispresent; wherein the step of processing comprises the steps of:passingthe color image through a hole filling filter; passing the filled colorimage through an erosion filter; operating on the eroded filled colorimage to define outlines around areas; selecting pixels within theoutlines by performing a logical NOT operation; and performing a logicalAND operation between the selected pixels and the filled color image. 3.The product of claim 1, wherein the genetic abnormality is human trisomy21.
 4. The product of claim 1, wherein the step of processing furtherincludes steps of:manually selecting a plurality of pixels within thebackground; determining color intensity value ranges corresponding tothe portion of the background; and identifying as the background thoseareas of the image having color intensity values within the rangesdetermined.
 5. A method of operating a computer to count occurrences ofa target substance in a cell containing sample which has been labeledwith a target-specific fluorophor, the method comprising:receiving adigitized color image of the fluorophor-labeled sample; obtaining acolor image of the fluorophor-labeled sample; separating objects ofinterest from background in the color image; measuring parameters of theobjects of interest so as to enumerate objects having specificcharacteristics; and analyzing the enumeration of objects with respectto a statistically expected enumeration to determine whether saidexpected enumeration associated with the target substance is present;wherein the step of separating comprises the steps of:passing the colorimage through a hole filling filter; passing the filled color imagethrough an erosion filter; operating on the eroded filled color image,to define outlines around areas; selecting pixels within the outlines byperforming a logical NOT operation; and performing a logical ANDoperation between the selected pixels and the filled color image.
 6. Themethod of claim 5, wherein the target substance is human chromosome 21.7. The method of claim 5, wherein the step of separating furtherincludes steps of:manually selecting a plurality of pixels within thebackground; determining color intensity value ranges corresponding tothe portion of the background; and identifying as the background thoseareas of the image having color intensity values within the rangesdetermined.
 8. A computer software product comprising:a computerreadable storage medium having fixed therein a sequence of computerinstructions directing a computer system to count occurrences of atarget substance in a cell-containing sample which has been labeled witha target-specific fluorophor, the instructions directing steps of:receiving a digitized color image of the fluorophor-labeled sample;obtaining a color image of the fluorophor-labeled sample; separatingobjects of interest from background in the color image; wherein the stepof separating comprises the steps of:passing the color image through ahole filling filter; passing the filled color image through an erosionfilter; operating on the eroded filled color image, to define outlinesaround areas; selecting pixels within the outlines by performing alogical NOT operation; and performing a logical AND operation betweenthe selected pixels and the filled color image measuring parameters ofthe objects of interest so as to enumerates object having specificcharacteristics; and analyzing the enumeration of objects with respectto a statistically expected enumeration to determine whether saidexpected enumeration of target substance is present.
 9. The product ofclaim 8, wherein the step of separating further includes stepsof:manually selecting a plurality of pixels within the background;determining color intensity value ranges corresponding to the portion ofthe background; and identifying as the background those areas of theimage having color intensity values within the ranges determined. 10.Apparatus for analyzing an image of a cell-containing sample which hasbeen labeled with a target-specific fluorophor associated with a geneticabnormality, comprising a computer system on which image processingsoftware executes; anda storage medium in which is fixed a sequence ofimage processing instructions including receiving a digitized colorimage of the fluorophor-labeled sample, obtaining a color image of thefluorophor-labeled sample, separating objects of interest frombackground in the color image, wherein the step of separating comprisesthe steps of:passing the color image through a hole filling filter;passing the filled color image through an erosion filter; operating onthe eroded filled color image, to define outlines around areas;selecting pixels within the outlines by performing a logical NOToperation; and performing a logical AND operation between the selectedpixels and the filled color image; measuring parameters of the objectsof interest so as to enumerate objects having specific characteristics,and analyzing the enumeration of objects with respect to a statisticallyexpected enumeration to determine presence of the genetic abnormality.11. The method of claim 2, wherein the genetic abnormality is in humanchromosome X, Y, 13, 18 or
 21. 12. The product of claim 1, wherein thegenetic abnormality is in human chromosome X, Y, 13, 18 or
 21. 13. Themethod of claim 5, wherein the target substance is human chromosome X,Y, 13, 18 or
 21. 14. The product of claim 8, wherein the targetsubstance is human chromosome X, Y, 13, 18 or
 21. 15. The apparatus ofclaim 10, wherein the genetic abnormality is in human chromosome X, Y,13, 18 or
 21. 16. The method of claim 2, wherein the genetic abnormalityis human trisomy
 21. 17. The product of claim 1, wherein the geneticabnormality is human trisomy
 21. 18. The method of claim 5, wherein thetarget substance is human chromosome
 21. 19. The product of claim 8,wherein the target substance is human chromosome
 21. 20. The apparatusof claim 10, wherein the genetic abnormality is human trisomy 21.